OxiSelect Superoxide Dismutase Activity Assay

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1 Product Manual OxiSelect Superoxide Dismutase Activity Assay Catalog Number STA assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures

2 Introduction Reactive oxygen species (ROS), such as superoxide (O 2 - ) and hydrogen peroxide (H 2 O 2 ), are constantly produced during metabolic processes in all living species. Under normal physiological conditions, cellular ROS generation is counterbalanced by the action of antioxidant enzymes and other redox molecules. However, excessive ROS accumulation will lead to cellular injury, such as damage to DNA, protein, and lipid membrane. Because of their potential harmful effects, excessive ROS must be promptly eliminated from the cells by a variety of antioxidant defense mechanisms. Superoxide dismutase (SOD), which catalyzes the dismutation of the superoxide anion into hydrogen peroxide and molecular oxygen, is one of the most important antioxidative enzymes. SOD enzymes are classified into three groups: cytosolic Cu/Zn-SOD, mitochondrial Mn-SOD, and extracellular Ec-SOD. Our OxiSelect Superoxide Dismutase Activity Assay uses a xanthine/xanthine oxidase (XOD) system to generate superoxide anions. The included chromagen produces a water-soluble formazan dye upon reduction by superoxide anions. The activity of SOD is determined as the inhibition of chromagen reduction (See Figure 1). The OxiSelect Superoxide Dismutase Activity Assay is a fast and reliable kit for the measurement of SOD activity from cell lysate, plasma, serum, tissue homogenates. Each kit provides sufficient reagents to perform up to 100 assays, including blanks, SOD standards and unknown protein samples. Test Principle Superoxide anions (O 2 -) are generated by a Xanthine/Xanthine Oxidase (XOD) system, and then detected with a Chromagen Solution. However, in the presence of SOD, these superoxide anion concentrations are reduced, yielding less colorimetric signal. Figure 1: Assay Principle 2

3 Related Products 1. STA-305: OxiSelect Nitrotyrosine ELISA Kit (96 Assays) 2. STA-310: OxiSelect Protein Carbonyl ELISA Kit (96 Assays) 3. STA-320: OxiSelect Oxidative DNA Damage ELISA (8-OHdG, 96 Assays) 4. STA-330: OxiSelect TBARS Assay Kit (MDA Quantitation) 5. STA-332: OxiSelect MDA Adduct ELISA Kit (96 Assays) Kit Components 1. SOD Standard (Part No ): One 100 µl vial provided at 5 Units/µL. Unit Definition: One unit will inhibit the rate of reduction of cytochrome c by 50% in a coupled system, using xanthine and xanthine oxidase, at ph 7.8 at 25 C in a 3.0 ml reaction volume. 2. Xanthine Solution (Part No ): One 550 µl vial. 3. Xanthine Oxidase Solution, 150X (Part No ): One 50 µl vial. 4. Chromagen Solution (Part No ): One 550 µl amber vial. 5. SOD Assay Buffer, 10X (Part No ): Two 1.5 ml vials. Materials Not Supplied well microtiter plate 2. 37ºC incubator or water bath µl to 1000 µl adjustable single channel micropipettes with disposable tips µl to 300 µl adjustable multichannel micropipette with disposable tips 5. Multichannel micropipette reservoir 6. Microplate reader capable of reading at 490 nm Storage Store kit components at -20ºC until their expiration dates. Avoid multiple freeze/thaws by aliquoting. Chromagen Solution is light sensitive and should be maintained in amber tubes. Preparation of Reagents 1X SOD Assay Buffer: Dilute one vial of 10X SOD Assay Buffer to 1X with deionized water. Mix to homogeneity. Keep the second vial of 10X SOD Assay Buffer undiluted. 1X Xanthine Oxidase Solution: Just prior to use, dilute the 150X Xanthine Oxidase Solution to 1X with 1X SOD Assay Buffer. Mix to homogeneity. Special Precautions Avoid the use of reducing agents, such as DTT, in the assay due to interference with the Chromagen Solution. 3

4 Assay Protocol 1. 1) Cytosolic fraction preparation: a) Suspension Cells- Centrifuge 3-6 x 10 6 cells at 700 x g for 2 minutes and discard supernatant. Wash cell pellet once with ice-cold PBS, centrifuge, and discard the supernatant. Resuspend cell pellet in 0.5 ml of cold 1X Lysis Buffer (10 mm Tris, ph 7.5, 150 mm NaCl, 0.1 mm EDTA, 0.5% Triton-100) and incubate on ice for 10 minutes. Centrifuge at x g for 10 minutes and collect the cell lysate supernatant. b) Adherent Cells- Wash 1-5 x 10 6 cells once with 10 ml ice-cold PBS per 100 mm dish. Place dish on ice. Add 1 ml of cold 1X Lysis Buffer (10 mm Tris, ph 7.5, 150 mm NaCl, 0.1 mm EDTA, 0.5% Triton-100) and incubate on ice for 10 minutes. Collect cells/debris with a rubber policeman. Transfer the cell extract to a microfuge tube and centrifuge at x g for 10 minutes. Collect the cell lysate supernatant. 2) Tissue lysate preparation: homogenize tissue sample in 5-10 ml of cold 1X Lysis Buffer (10 mm Tris, ph 7.5, 150 mm NaCl, 0.1 mm EDTA, 0.5% Triton-100) per gram tissue. Centrifuge at x g for 10 minutes and collect the tissue lysate supernatant. 2. Prepare samples including a blank in a 96-well microtiter plate according to the below table. Allow pre-incubation time if inhibitor is used. Component Blank Sample SOD Sample 0 µl X µl Inhibitor (optional) 0 µl Y µl Xanthine Solution 5 µl 5 µl Chromagen Solution 5 µl 5 µl 10X SOD Assay Buffer 10 µl 10 µl DI Water 70 µl 70-(X+Y) µl Total 90 µl 90 µl 3. Finally, add 10 µl of pre-diluted 1X Xanthine Oxidase Solution (see Preparation of Reagents) to each well. Mix well and incubate for 1 hour at 37ºC. 4. Read absorbance at 490 nm on a microplate reader. Preparation of SOD Standards (Optional) 1. Thaw SOD Standard at 4ºC. 2. Freshly prepare a dilution series (1:4 is suggested) of SOD Standard in the concentration range of 5 Units/µL 0.61 mu/µl by diluting the SOD Standard in 1X Assay Buffer (see Preparation of Reagents). 3. Transfer 10 µl of each dilution to a 96-well microtiter plate, including a 1X Assay Buffer blank. 4

5 4. Prepare the following master mixture, adjusting for the required number of wells. Component Volume per well Xanthine Solution 5 µl Chromagen Solution 5 µl 10X SOD Assay Buffer 10 µl DI Water 60 µl Total 80 µl 5. Transfer 80 µl of the above master mixture to each well. 6. Finally, add 10 µl of pre-diluted 1X Xanthine Oxidase Solution (see Preparation of Reagents) to each well. Mix well and incubate for 1 hour at 37ºC. 7. Read absorbance at 490 nm on a microplate reader. Example of Results The following figures demonstrate typical OxiSelect SOD Activity Assay results. One should use the data below for reference only. This data should not be used to interpret actual results. SOD Activity (inhibition %) = (OD blank -OD sample )/( OD blank ) x 100 OD 490nm SOD (Units) Inhibition (%) SOD (Units) Figure 2: SOD Activity Assay Standard Curve. 5

6 References 1. Lepock, J. R., Arnold, L. D., Torrie, B. H., Andrews, B., and Kruuv, J. (1985) Arch. Biochem. Biophys. 241, Lepock, J. R., Frey, H. E., and Hallewell, R. A. (1990) J. Biol. Chem. 265, Valentine, J. S., and Hart, P. J. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, Zelko, I. N., Mariani, T. J., and Folz, R. J. (2002) Free Radic. Biol. Med. 33, Warranty These products are warranted to perform as described in their labeling and in Cell Biolabs literature when used in accordance with their instructions. THERE ARE NO WARRANTIES THAT EXTEND BEYOND THIS EXPRESSED WARRANTY AND CELL BIOLABS DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE. CELL BIOLABS s sole obligation and purchaser s exclusive remedy for breach of this warranty shall be, at the option of CELL BIOLABS, to repair or replace the products. In no event shall CELL BIOLABS be liable for any proximate, incidental or consequential damages in connection with the products. Contact Information Cell Biolabs, Inc Arjons Drive San Diego, CA Worldwide: USA Toll-Free: CBL : Cell Biolabs, Inc. - All rights reserved. No part of these works may be reproduced in any form without permissions in writing. 6

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